Methods for isolating crystalline form i of 5-azacytidine

ABSTRACT

The invention includes methods for isolating crystalline Form I of 5-azacytidine substantially free of other forms, wherein 5-azacytidine is represented by the formula: 
     
       
         
         
             
             
         
       
     
     The invention also includes pharmaceutical compositions comprising Form I of 5-azacytidine.

FIELD OF THE INVENTION

The invention relates to the isolation of crystalline polymorphic Form Iof 5-azacytidine (also known as azacitidine and4-amino-1-β-D-ribofuranosyl-S-triazin-2(1H)-one). 5-azacytidine may beused in the treatment of disease, including the treatment ofmyelodysplastic syndromes (MDS).

BACKGROUND OF THE INVENTION

Polymorphs exist as two or more crystalline phases that have differentarrangements and/or different conformations of the molecule in a crystallattice. When a solvent molecule(s) is contained within the crystallattice the resulting crystal is called a pseudopolymorph, or solvate.If the solvent molecule(s) within the crystal structure is a watermolecule, then the pseudopolymorph/solvate is called a hydrate. Thepolymorphic and pseudopolymorphic solids display different physicalproperties, including those due to packing, and various thermodynamic,spectroscopic, interfacial and mechanical properties (See H. Brittain,Polymorphism in Pharmaceutical Solids, Marcel Dekker, New York, N.Y.,1999, pp. 1-2). Polymorphic and pseudopolymorphic forms of the drugsubstance (also known as the “active pharmaceutical ingredient” (API)),as administered by itself or formulated as a drug product (also known asthe final or finished dosage form, or as the pharmaceutical composition)are well known and may affect, for example, the solubility, stability,flowability, fractability, and compressibility of drug substances andthe safety and efficacy of drug products, (see, e.g., Knapman, K ModemDrug Discoveries, March 2000: 53).

5-azacytidine (also known as azacitidine and4-amino-1-β-D-ribofuranosyl-S-triazin-2(1H)-one; Nation Service Centerdesignation NSC-102816; CAS Registry Number 320-67-2) has undergoneNCI-sponsored trials for the treatment of myelodysplastic syndromes(MDS). See Komblith et al., J. Clin. Oncol. 20(10): 2441-2452 (2002) andSilverman et al., J. Clin. Oncol. 20(10): 2429-2440 (2002).5-azacytidine may be defined as having a formula of C₈H₁₂N₄O₅, amolecular weight of 244.20 and a structure of:

In the United States patent application entitled “Forms of5-azacytidine,” Attorney Docket No. PHAR.02, filed Mar. 17, 2003 andincorporated herein by reference in its entirety, eight differentpolymorphic and pseudopolymorphic forms of 5-azacytidine (Forms I-VIII),in addition to an amorphous form, are described. Forms each havecharacteristic X-Ray Powder Diffraction (XRPD) patterns and are easilydistinguished from one another using XRPD.

5-azacytidine drug substance used in the previous clinical trials hastypically been synthesized from 5-azacytosine and1,2,3,5,-tetra-O-acetyl-β-D-ribofuranose by the method presented inExample 1. The last step of this method is a recrystallization of thecrude synthesis product from a methanol/DMSO co-solvent system.Specifically, the crude synthesis product is dissolved in DMSO(preheated to about 90° C.), and then methanol is added to the DMSOsolution. The product is collected by vacuum filtration and allowed toair dry.

In Attorney Docket No. PHAR.02 (supra), it is demonstrated that thisprior art method for the recrystallization of the crude synthesisproduct does not control for the polymorphic forms of 5-azacytidine.Specifically, the prior art recrystallization procedure produces eitherForm I substantially free of other forms, or a Form I/II mixed phasei.e. a solid material in which 5-azacytidine is present in a mixed phaseof both polymorphic Form I and polymorphic Form II. Thus, the prior artprocedures do not allow one to reliably target Form I as the singlepolymorphic form in the drug substance. The present invention providesmethods that allow one to recrystallize 5-azacytidine as polymorphicForm I robustly and reproducibly.

SUMMARY OF THE INVENTION

The present invention provides methods for robustly and reproduciblyisolating 5-azacytidine as polymorphic Form I substantially free ofother forms. The methods involve recrystallizing dissolved 5-azacytidinefrom a primary solvent/co-solvent mixture and then collecting theresultant crystals. The invention also provides pharmaceuticalcompositions comprising Form I of 5-azacytidine together with apharmaceutically acceptable excipient, diluent, or carrier.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Polymorphic Form I of5-azacytidine

Form I of 5-azacytidine is described fully in Attorney Docket No.PHAR.02 (see supra), and such descriptions are incorporated by referenceherein. Table I provides the most prominent 2θ angles, d-spacing andrelative intensities for Form I observed using X-Ray Powder Diffraction(XRPD) performed according the method of Example 4:

TABLE 1 5-azacytidine Form I - the most prominent 2θ angles, d-spacingand relative intensities (Cu Kα radiation) 2θ Angle (°) d-spacing (Å)Relative Intensity 12.182 7.260 39.1 13.024 6.792 44.1 14.399 6.146 31.516.470 5.378 27.1 18.627 4.760 16.0 19.049 4.655 35.9 20.182 4.396 37.021.329 4.162 12.4 23.033 3.858 100.0 23.872 3.724 28.0 26.863 3.316 10.827.135 3.284 51.5 29.277 3.048 25.6 29.591 3.016 11.5 30.369 2.941 10.832.072 2.788 13.4Isolation of Polymorphic Form I of 5-azacytidine by Recrystallization

Form I of 5-azacytidine may be reproducibly isolated substantially freeof other forms by recrystallizing dissolved 5-azacytidine and collectingthe resultant crystals. Specifically, 5-azacytidine is first dissolvedcompletely in at least one suitable primary solvent, preferably a polarsolvent, more preferably a polar aprotic solvent. Suitable polar aproticsolvents include, but are not limited to, dimethylformamide (DMF),dimethylacetamide (DMA), dimethylsulfoxide (DMSO), andN-methylpyrrolidinone (NMP). The most preferred polar aprotic solvent isDMSO. Mixtures of two or more primary solvents are also contemplated fordissolving the 5-azacytidine, for example a mixture of DMSO and DMF.

The 5-azacytidine used to form the solution may be synthesized by anyprocedure known in the art; an exemplary prior art synthesis scheme isprovided in Example 1. Any polymorphic or pseudopolymorphic form(s) of5-azacytidine, including mixed phases, may be used to form the solution.Amorphous 5-azacytidine may also be used to form the solution. It ispreferred, but not required, that the primary solvent is preheated to anelevated temperature in order to ensure that the 5-azacytidine isdissolved completely. An especially preferred primary solvent isdimethyl sulfoxide, (DMSO), most preferably preheated to a temperaturein the range of about 40° C. to about 90° C.

Following solvation of the 5-azacytidine in the primary solvent, atleast one co-solvent is added to the solution of 5-azacytidine. Suitableco-solvents include C₂-C₅ alcohols (which term hereinafter refers toC₂-C₅ alcohols that are independently: branched or unbranched,substituted or unsubstituted), aliphatic ketones (which term hereinafterrefers to aliphatic ketones that are indepedently: branched orunbranched, substituted or unsubstituted), and alkyl cyanides (whichterm hereinafter refers to alkyl cyanides that are independently:branched or unbranched, substituted or unsubstituted). Preferred C₂-C₅alcohols, aliphatic ketones, and alkyl cyanides, along with othersuitable solvents, are listed below as Class 2 (solvents to be limited)and Class 3 (solvents of low toxic potential) per the InternationalConference on Harmonization's (ICH) Guideline for Residual Solvents,July 1997). The use of mixtures of two or more of any of theaforementioned co-solvents is also included within the scope of theinvention.

Class 2

Acetonitrile

Chlorobenzene

Cyclohexane

1,2-Dichloroethene

Dichloromethane

1,2-Dimethoxyethane

N,N-Dimethylformamide

N,N-Dimethylacetamide

1,4-Dioxane

2-Ethoxyethanol

Ethyleneglycol

Formamide

2-Methoxyethanol

Methylbutyl ketone

Methylcyclohexane

Nitromethane

Pyridine

Sulfolane

Tetralin

1,1,2-Trichloroethene

Class 3

1-Butanol

1-Pentanol

1 -Propanol

2-Butanol

2-Methyl-1-propanol

2-Propanol (isopropyl alcohol)

3-Methyl-1-butanol

Acetone

Anisole

Butyl acetate

Cumene

Ethanol

Ethyl acetate

Ethyl ether

Ethyl formate

Isobutyl acetate

Isopropyl acetate

Methyl acetate

Methylethyl ketone

Methylisobutyl ketone

Propyl acetate

tert-Butylmethyl ether

Tetrahydrofuran

It is preferred, but not required, that the co-solvents are preheatedbefore mixing with the primary solvent, preferably to a temperaturebelow the temperature at which a substantial portion of the co-solventwould boil, most preferably to about 50° C. It is also preferred, butnot required, that the co-solvent(s) is added gradually to the primarysolvent(s).

Following mixing, the primary solvent(s)/co-solvent(s) mixture is thenequilibrated at different temperatures in order to promote either a slowrecrystallization or a fast recrystallization of Form I of5-azacytidine, as described below.

By slow recrystallization is meant that the co-solvent/DMSO solution isallowed to equilibrate at a temperature in the range from about 0° C. toabout 40° C., preferably in the range of about 15° C. to about 30° C.,and most preferably at about ambient temperature. Slow recrystallizationof Form I of 5-azacytidine is preferably performed using C₂-C₅ alcohols,aliphatic ketones, or alkyl cyanides as the co-solvent. More preferably,slow recrystallization is performed with Class 3 C₂-C₅ alcohols, Class 3aliphatic ketones, or acetonitrile (Class 2). The most preferred Class 3C₂-C₅ alcohols are ethanol, isopropyl alcohol, and 1-propanol, and themost preferred Class 3 aliphatic ketone is methylethyl ketone.

By fast recrystallization is meant that the co-solvent solution isallowed to equilibrate at a temperature of below 0° C., preferably belowabout −10° C., and most preferably at about −20° C. Fastrecrystallization of Form I of 5-azacytidine is preferably performedwith a C₃-C₅ alcohol (which term hereinafter refers to C₃-C₅ alcoholswhich are independently: branched or unbranched, substituted orunsubstituted) or an alkyl cyanide as the co-solvent. More preferablythe C₃-C₅ alcohol is a Class 3 solvent, and the alkyl cyanide isacetonitrile. The most preferred Class 3 C₃-C₅ alcohols are isopropylalcohol (2-propanol) and 1-propanol.

Non-limiting examples of protocols for the recrystallization of Form Iaccording to the methods described herein are provided in Examples 2(slow recrystallization with DMSO as the primary solvent and ethanol,isopropyl alcohol, acetonitrile, or methylethyl ketone as theco-solvent) and 3 (fast recrystallization with DMSO as the primarysolvent, and isopropyl alcohol or acetonitrile as the co-solvent) below.

Following recrystallization, the Form I of 5-azacytidine crystals may beisolated from the co-solvent mixture by any suitable method known in theart. Preferably, the Form I crystals are isolated using vacuumfiltration through a suitable filter medium or by centrifugation.

Using the novel methods provided herein, it is possible for the firsttime to target Form I of 5-azacytidine as the drug substancereproducibly and robustly. In particular, isopropyl alcohol andacetonitrile reliably produce Form I independent of cooling rate (eitherslow recrystallization or fast recrystallization) and are preferred asthe recrystallization co-solvents to recover Form I. Most preferably,Form I is isolated using isopropyl alcohol as the co-solvent sinceisopropyl alcohol carries a Class 3 risk classification (solvent of lowtoxic potential), whereas acetonitrile carries a Class 2 riskclassification (solvent to be limited). The use of the DMSO/isopropylalcohol system allows Form I of 5-azacytidine to be reliably recoveredfor the first time from solvents of low toxic potential withoutrequiring control over the rate of recrystallation. In the mostpreferred embodiment, Form I of 5-azacytidine may be recovered simply bydissolving 5-azacytidine in DMSO (preferably heated to a temperature inthe range of about 40° C. to about 90° C. prior to the addition of5-azacytidine), adding isopropyl alcohol, and allowing the resultingsolvent mixture to equilibrate at about ambient temperature.

In some embodiments of the invention, Form I of 5-azacytidine may berecovered from a primary solvent(s)/co-solvent(s) mixture by “seeding”with a small amount of Form I of 5-azacytidine either prior to, orduring, the addition of the co-solvent(s). By seeding with Form I, it ispossible to expand the list of suitable co-solvents and co-solventclasses beyond those listed above. For example, it is known thatrecrystallization from the DMSO/methanol system produces either Form I,or a Form VII mixed phase (see Example 1). If a small amount of Form Iis added to the solution of 5-azacytidine in DMSO prior to addition ofthe methanol co-solvent, or is added during the addition of the methanolco-solvent, then Form I of 5-azacytidine may be reliably isolated.

By allowing the isolation of a single polymorphic form, one skilled inthe art will appreciate that the present invention allows for the firsttime the production of 5-azacytidine drug substance with uniform andconsistent properties from batch to batch, which properties include butare not limited to solubility and dissolution rate. In turn, this allowsone to provide 5-azacytidine drug product (see below) which also hasuniform and consistent properties from batch to batch.

Pharmaceutical Formulations

For the most effective administration of drug substance of the presentinvention, it is preferred to prepare a pharmaceutical formulation (alsoknown as the “drug product” or “pharmaceutical composition”) preferablyin unit dose form, comprising one or more of the 5-azacytidinepolymorphs of the present invention and one or more pharmaceuticallyacceptable carrier, diluent, or excipient. Most preferably, Form I5-azacytidine prepared according to the methods provided herein is usedto prepare the pharmaceutical formulation.

Such pharmaceutical formulation may, without being limited by theteachings set forth herein, include a solid form of the presentinvention which is blended with at least one pharmaceutically acceptableexcipient, diluted by an excipient or enclosed within such a carrierthat can be in the form of a capsule, sachet, tablet, buccal, lozenge,paper, or other container. When the excipient serves as a diluent, itmay be a solid, semi-solid, or liquid material which acts as a vehicle,carrier, or medium for the 5-azacytidine polymorph(s). Thus, theformulations can be in the form of tablets, pills, powders, elixirs,suspensions, emulsions, solutions, syrups, capsules (such as, forexample, soft and hard gelatin capsules), suppositories, sterileinjectable solutions, and sterile packaged powders.

Examples of suitable excipients include, but are not limited to,starches, gum arabic, calcium silicate, microcrystalline cellulose,polyvinylpyrrolidone, cellulose, water, syrup, and methyl cellulose. Theformulations can additionally include lubricating agents such as, forexample, talc, magnesium stearate and mineral oil; wetting agents;emulsifying and suspending agents; preserving agents such as methyl- andpropyl-hydroxybenzoates; sweetening agents; or flavoring agents.Polyols, buffers, and inert fillers may also be used. Examples ofpolyols include, but are not limited to: mannitol, sorbitol, xylitol,sucrose, maltose, glucose, lactose, dextrose, and the like. Suitablebuffers encompass, but are not limited to, phosphate, citrate, tartrate,succinate, and the like. Other inert fillers which may be used encompassthose which are known in the art and are useful in the manufacture ofvarious dosage forms. If desired, the solid pharmaceutical compositionsmay include other components such as bulling agents and/or granulatingagents, and the like. The compositions of the invention can beformulated so as to provide quick, sustained, controlled, or delayedrelease of the drug substance after administration to the patient byemploying procedures well known in the art.

In certain embodiments of the invention, the 5-azacytidine polymorph(s)may made into the form of dosage units for oral administration. The5-azacytidine polymorph(s) may be mixed with a solid, pulverant carriersuch as, for example, lactose, saccharose, sorbitol, mannitol, starch,amylopectin, cellulose derivatives or gelatin, as well as with anantifriction agent such as for example, magnesium stearate, calciumstearate, and polyethylene glycol waxes. The mixture is then pressedinto tablets or filled into capsules. If coated tablets, capsules, orpulvules are desired, such tablets, capsules, or pulvules may be coatedwith a concentrated solution of sugar, which may contain gum arabic,gelatin, talc, titanium dioxide, or with a lacquer dissolved in thevolatile organic solvent or mixture of solvents. To this coating,various dyes may be added in order to distinguish among tablets withdifferent active compounds or with different amounts of the activecompound present.

Soft gelatin capsules may be prepared in which capsules contain amixture of the 5-azacytidine polymorph(s) and vegetable oil ornon-aqueous, water miscible materials such as, for example, polyethyleneglycol and the like. Hard gelatin capsules may contain granules orpowder of the 5-azacytidine polymorph in combination with a solid,pulverulent carrier, such as, for example, lactose, saccharose,sorbitol, mannitol, potato starch, corn starch, amylopectin, cellulosederivatives, or gelatin.

Tablets for oral use are typically prepared in the following manner,although other techniques may he employed. The solid substances aregently ground or sieved to a desired particle size, and a binding agentis homogenized and suspended in a suitable solvent. The 5-azacytidinepolymorph(s) and auxiliary agents are mixed with the binding agentsolution. The resulting mixture is moistened to form a uniformsuspension. The moistening typically causes the particles to aggregateslightly, and the resulting mass is gently pressed through a stainlesssteel sieve having a desired size. The layers of the mixture are thendried in controlled drying units for a pre-determined length of time toachieve a desired particle size and consistency. The granules of thedried mixture are gently sieved to remove any powder. To this mixture,disintegrating, anti-friction, and anti-adhesive agents are added.Finally, the mixture is pressed into tablets using a machine with theappropriate punches and dies to obtain the desired tablet size.

In the event that the above formulations are to be used for parenteraladministration, such a formulation typically comprises sterile, aqueousand non-aqueous injection solutions comprising one or more 5-azacytidinepolymorphs for which preparations are preferably isotonic with the bloodof the intended recipient. These preparations may contain anti-oxidants,buffers, bacteriostats, and solute; which render the formulationisotonic with the blood of the intended recipient. Aqueous andnon-aqueous suspensions may include suspending agents and thickeningagents. The formulations may be present in unit-dose or multi-dosecontainers, for example, sealed ampules and vials. Extemporaneousinjection solutions and suspensions may be prepared from sterilepowders, granules, and tablets of the kind previously described.

Liquid preparations for oral administration are prepared in the form ofsolutions, syrups, or suspensions with the latter two forms containing,for example, 5-azacytidine polymorph(s), sugar, and a mixture ofethanol, water, glycerol, and propylene glycol. If desired, such liquidpreparations contain coloring agents, flavoring agents, and saccharin.Thickening agents such as carboxymethylcellulose may also be used.

As such, the pharmaceutical formulations of the present invention arepreferably prepared in a unit dosage form, each dosage unit containingfrom about 5 mg to about 200 mg, more usually about 100 mg of the5-azacytidine polymorph(s). In liquid form, dosage unit contains fromabout 5 to about 200 mg, more usually about 100 mg of the 5-azacytidinepolymorph(s). The term “unit dosage form” refers to physically discreteunits suitable as unitary dosages for human subjects/patients or othermammals, each unit containing a predetermined quantity of the5-azacytidine polymorph calculated to produce the desired therapeuticeffect, in association with preferably, at least one pharmaceuticallyacceptable carrier, diluent, or excipient.

The following examples are provided for illustrative purposes only, andare not to be construed as limiting the scope of the claims in any way.

EXAMPLES Example 1

Prior Art Procedure for Synthesis and Recrystallization of 5-azacytidineDrug Substance

5-azacytidine may be synthesized using commercially available5-azacytosine and 1,2,3,5-tetra-O-acetyl-β-D-ribofuranose (RTA)according to the following pathway:

The crude synthesis product is dissolved in DMSO (preheated to about 90°C.), and then methanol is added to the DMSO solution. The co-solventmixture is equilibrated at approximately −20° C. to allow 5-azacytidinecrystal formation. The product is collected by vacuum filtration andallowed to air dry.

Example 2

Form I of 5-azacytidine: Slow Recrystallization of 5-azacytidine fromCo-Solvent Systems

Approximately 250 mg of 5-azacytidine was dissolved with approximately 5ml of dimethyl sulfoxide (DMSO), preheated to approximately 90° C., inseparate 100-mL beakers. The solids were allowed to dissolve to a clearsolution. Approximately 45 mL of ethanol, isopropyl alcohol,acetonitrile, or methyl ethyl ketone co-solvent, preheated toapproximately 50° C., was added to the solution and the resultantsolution was mixed. The solution was covered and allowed to equilibrateat ambient conditions. The product was collected by vacuum filtrationusing a Buchner funnel.

Example 3

Form I of 5-azacytidine: Fast Recrystallization of 5-azacytidine fromCo-Solvent Systems

Approximately 250 mg of 5-azacytidine was dissolved with approximately 5mL of DMSO, preheated to approximately 90° C., in separate 100-mlbeakers. The solids were allowed to dissolve to a clear solution.Approximately 45 mL of isopropyl alcohol or acetonitrile co-solvent,preheated to approximately 50° C., was added to the solution and theresultant solution was mixed. The solution was covered and placed in afreezer to equilibrate at approximately −20° C. to allow crystalformation. Solutions were removed from the freezer after crystalformation. The product was collected by vacuum filtration using aBuchner funnel.

Example 4

X-Ray Powder Diffraction of Recrystallized 5-azacytidine

X-ray powder diffraction (XRPD) patterns for each sample were obtainedon a Scintag XDS 2000 or a Scintag X₂ θ/θ diffractometer operating withcopper radiation at 45 kV and 40 mA using a Kevex Psi Peltier-cooledsilicon detector or a Thermo ARL Peltier-cooled solid state detector.Source slits of 2 or 4 mm and detector slits of 0.5 or 0.3 mm were usedfor data collection. Recrystallized material was gently milled forapproximately one minute using an agate mortar and pestle. Samples wereplaced in a stainless steel or silicon sample holder and leveled using aglass microscope slide. Powder diffraction patterns of the samples wereobtained from 2 to 42° 2θ at 1°/minute. Calibration of the X₂diffractometer is verified annually using a silicon powder standard.

XRPD performed according to this method revealed that the Form I of5-azacytidine was isolated in Example 2 by slow recrystallization usingeither ethanol, isopropyl alcohol, acetonitrile, or methyl ethyl ketoneas the co-solvent, and in Example 3 by fast recrystallization usingisopropyl alcohol or acetonitrile as the co-solvent. The resultsindicate that Form I of 5-azacytidine may be reliably recovered from theDMSO/isopropyl alcohol and DMSO/acetonitrile solvent systems withoutcontrol of the rate of recrystallization.

1-36. (canceled)
 37. A method for isolating crystalline Form I of 5-azacytidine substantially free of other forms, the method comprising: recrystallizing 5-azacytidine from a solvent mixture comprising dimethylsulfoxide and isopropyl alcohol; and isolating the recrystallized 5-azacytidine.
 38. A method for isolating crystalline Form I of 5-azacytidine substantially free of other forms, the method comprising: recrystallizing 5-azacytidine from a solvent mixture comprising dimethylsulfoxide and 1-propanol; and isolating the recrystallized 5-azacytidine.
 39. A method for isolating crystalline Form I of 5-azacytidine substantially free of other forms, the method comprising: recrystallizing 5-azacytidine from a solvent mixture comprising dimethylsulfoxide and methanol by seeding said solvent mixture with Form I of 5-azacytidine; and isolating the recrystallized 5-azacytidine. 